The 26S proteasome performs regulated proteolysis in eukaryotes and has emerged as a major therapeutic target. It is composed of a 20S catalytic core particle (CP) where protein degradation occurs, that is capped at either end by a 19S regulatory particle (RP). Proteasome substrates are distinguished by post-translational modification with ubiquitin chains, which bind directly to receptor sites in the RP. A major goal of our section is to understand how the proteasome recognizes substrates and becomes committed to substrate proteolysis. We aim to use this knowledge to design new methods that target specific components of the ubiquitin-proteasome pathway. In collaboration with extramural researchers, we established Rpn1 and Rpn13 as substrate receptors in the proteasome that can directly interact with ubiquitin or ubiquitin-like domains of shuttle factors that contain ubiquitin-associated domains. We used NMR spectroscopy to solve the structure of the three major substrate receptor sites in the RP (Rpn1, Rpn10 and Rpn13) complexed with ubiquitin chains. This work is being extended to evaluate the specific functional roles of Rpn1, Rpn10 and Rpn13, both by biophysical methods and cell biology techniques. In addition, we are further defining proteasome structure at atomic level resolution. In a related collaborative project, we found that a class of bis-benzylidine piperidone derivatives trigger effects similar to proteasome inhibition, including restriction of tumor growth in mouse xenograft models. In contrast to the FDA-approved inhibitors, this class of small molecules (exemplified by RA190) do not inhibit the proteasome CP and instead covalently attach to hRpn13, which we have found is required for RA190 sensitivity. We are actively pursuing the mechanistic relationship between RA190 and hRpn13. Altogether, this project is providing fundamental information on how the proteasome recognizes ubiquitinated substrates at atomic level resolution, as well as the therapeutic potential of targeting alternative sites in the proteasome.